The lipid-rich cell wall is a defining feature of Mycobacterium species. Individual cell wall components affect diverse mycobacterial phenotypes including colony morphology, biofilm formation, antibiotic resistance, and virulence. In this study, we describe a transposon insertion mutant of Mycobacterium smegmatis mc 2 155 that exhibits altered colony morphology and defects in biofilm formation. The mutation was localized to the lsr2 gene. First identified as an immunodominant T-cell antigen of Mycobacterium leprae, lsr2 orthologs have been identified in all sequenced mycobacterial genomes, and homologs are found in many actinomycetes. Although its precise function remains unknown, localization experiments indicate that Lsr2 is a cytosolic protein, and cross-linking experiments demonstrate that it exists as a dimer. Characterization of cell wall lipid components reveals that the M. smegmatis lsr2 mutant lacks two previously unidentified apolar lipids. Characterization by mass spectrometry and thin-layer chromatography indicate that these two apolar lipids are novel mycolatecontaining compounds, called mycolyl-diacylglycerols (MDAGs), in which a mycolic acid (␣-or ␣-mycolate) molecule is esterified to a glycerol. Upon complementation with an intact lsr2 gene, the mutant reverts to the parental phenotypes and MDAG production is restored. This study demonstrates that due to its impact on the biosynthesis of the hydrophobic MDAGs, Lsr2 plays an important role in the colony morphology and biofilm formation of M. smegmatis.The cell wall is a defining feature of mycobacteria. This complex, lipid-rich, hydrophobic structure is responsible for the acid-fast staining properties, distinctive colony morphology, and innate antibiotic resistance of Mycobacterium species (12,25,28). Among pathogens, including Mycobacterium tuberculosis, the causative agent of tuberculosis, cell wall components contribute to virulence, persistence within macrophages, and modulation of the host immune response (16, 45).The cell wall forms an asymmetric lipid bilayer (25,28). The inner leaflet is composed of mycolic acids that are covalently bound to arabinogalactan, which is further linked to peptidoglycan via a phosphodiester bridge (12). The outer leaflet contains a variety of lipid components (26,28). In total, lipids comprise 60% (wt/wt) of the cell wall (12, 25). In addition to mycolic acids, various types of complex lipids are present in the cell wall. These include lipoglycans (e.g., lipoarabinomannan [LAM]), trehalose-containing glycolipids, phthiocerol dimycocerosates, phenolic glycolipids, and glycopeptidolipids (GPLs) (12, 25). The distribution of these lipids varies among mycobacterial species (12). Triacylglycerols (TAGs) are also present in the mycobacterial cell wall (35) and are thought to fill the gap between the meromycolate arm and the shorter ␣-chain of mycolic acids (28). Different lipids appear to have different roles. For example, LAM from M. tuberculosis, but not the structurally distinct LAM of nonpathogenic mycobacteria,...

SummaryLipooligosaccharides (LOSs) are antigenic glycolipids that are present in some species of Mycobacterium including the Canetti strain of M. tuberculosis. The core LOS structures from several mycobacterial organisms have been established, but the biosynthetic pathways of LOSs remain unknown. In this study, we describe two transposon insertion mutants of M. marinum that exhibit altered colony morphology. Cell wall analysis reveals that the MRS1271 mutant is defective in the synthesis of LOS-II, whereas the MRS1178 mutant accumulates an intermediate between LOS-I and -II. The genetic lesions were localized to two genes, MM2309 and MM2332. MM2309 encodes a UDP-glucose dehydrogenase that is involved in the synthesis of D-xylose. MM2332 is predicted to encode a decarboxylase. These two genes and a previously identified losA gene are localized in a gene cluster likely to be involved in the biosynthesis of LOSs. Our results also show that LOSs play an important role in sliding motility, biofilm formation, and infection of host macrophages. Taken together, our studies have identified, for the first time, a LOS biosynthetic locus. This is an important step in assessing the differential distribution of LOSs among Mycobacterium species and understanding the role of LOSs in mycobacterial virulence.

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes including cell wall biosynthesis and antibiotic resistance. However, the underlying molecular mechanisms remain unknown. In this article, we performed biochemical studies of Lsr2–DNA interactions and structure–function analysis of Lsr2. Analysis by atomic force microscopy revealed that Lsr2 has the ability to bridge distant DNA segments, suggesting that Lsr2 plays a role in the overall organization and compactness of the nucleoid. Mutational analysis identified critical residues and selection of dominant negative mutants demonstrated that both DNA binding and protein oligomerization are essential for the normal functions of Lsr2 in vivo. These results provide strong evidence that Lsr2 is a DNA bridging protein, which represents the first identification of such proteins in bacteria phylogenetically distant from the Enterobacteriaceae. DNA bridging by Lsr2 also provides a mechanism of transcriptional regulation by Lsr2.

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The behavior of water droplets located on graphene in the presence of various external electric fields (E-fields) is investigated using classical molecular dynamics (MD) simulations. We explore the effect of E-field on mass density distribution, water polarization as well as hydrogen bonds (H-bonds) to gain insight into the wetting properties of water droplets on graphene and their interfacial structure under uniform E-fields. The MD simulation results reveal that the equilibrium water droplets present a hemispherical, a conical and an ordered cylindrical shape with the increase of external E-field intensity. Accompanied by the shape variation of water droplets, the dipole orientation of water molecules experiences a remarkable change from a disordered state to an ordered state because of the polarization of water molecules induced by static E-field. The distinct two peaks in mass density and H-bond distribution profiles demonstrate that water has a layering structure in the interfacial region, which sensitively depends on the strong E-field (>0.8 V nm(-1)). In addition, when the external E-field is parallel to the substrate, the E-field would make the contact angle of the water droplets become small and increase its wettability. Our findings provide the possibility to control the structure and wetting properties of water on graphene by tuning the direction and intensity of external E-field which is of importance for relevant industrial processes on the solid surface.

Mycobacteria are naturally resistant to most common antibiotics and chemotherapeutic agents. The underlying molecular mechanisms are not fully understood. In this paper, we describe a hypersensitive mutant of Mycobacterium smegmatis, MS 2-39, which was isolated by screening for transposon insertion mutants of M. smegmatis mc 2 155 that exhibit increased sensitivity to rifampin, erythromycin, or novobiocin. The mutant MS 2-39 exhibited increased sensitivity to all three of the above mentioned antibiotics as well as fusidic acid, but its sensitivity to other antibiotics, including isoniazid, ethambutol, streptomycin, chloramphenicol, norfloxacin, tetracycline, and ␤-lactams, remained unchanged. Uptake experiment with hydrophobic agents and cell wall lipid analysis suggest that the mutant cell wall is normal. The transposon insertion was localized within the asnB gene, which is predicted to encode a glutamine-dependent asparagine synthetase. Transformation of the mutant with wild-type asnB of mc 2 155 or asnB of Mycobacterium tuberculosis complemented the drug sensitivity phenotype. These results suggest that AsnB plays a role in the natural resistance of mycobacteria.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for 2 million deaths and 10 million new infections each year. Clinical treatments of TB and other mycobacterial infections are difficult because mycobacteria are naturally resistant to most commonly used antibiotics and chemotherapeutic agents (14, 17). For instance, among the commonly used antibiotics against M. tuberculosis, the only ones found to be effective are rifampin and streptomycin, which are routinely used, in combination with isoniazid, pyrazinamide, and ethambutol, for chemotherapy of TB. Infections caused by nontuberculous mycobacteria such as the Mycobacterium avium complex (MAC) are increasingly common in immunocompromised individuals, which are often more difficult to treat because these organisms are resistant to standard anti-TB drugs including isoniazid and rifampin (14,17).The natural resistance of mycobacteria is caused primarily by the impermeability of the mycobacterial cell wall (6,14,17). The cell wall forms an asymmetric lipid bilayer with mycolic acids in the inner leaflet and extractable complex lipids in the outer leaflet (17,20). Mycolic acids are of extraordinary length and are highly saturated; as such, the mycolic acid-containing layer has extremely low fluidity, which forms a strong permeability barrier and contributes to the broad resistance (17,18,20). The natural resistance of mycobacteria also involves active efflux processes mediated by various transport systems. Effluxmediated resistances have been reported for fluoroquinolones (21, 28), tetracyclines (9, 27), isoniazid, ethambutol (7), pyrazinamide (33), erythromycin, and rifamycines (16). These efflux systems, however, often confer only low levels of resistance.In an attempt to better understand the mechanisms involved in the natural resistance of mycobacteria to antibiotics,...

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